It is generally accepted that an increase in intracellular [Ca2+] is responsible for regulating smooth muscle contraction. Increasing evidence suggests that the Ctimecourse and magnitude of Ca2+ signals generated in response to a wide range of stimuli are subject to feedback. Recent results suggest that protein kinases play a role in such feedback modulation of Ca2+ signalling. Activation of protein kinase C has recently been hypothesized to be an important regulatory component of the agonist stimulated responses of smooth muscle. Although the mechanism by which protein kinase C modulates smooth muscle contraction is not known, it has been proposed that protein kinase C is involved in the regulation of Ca2+ homeostasis of cells so as to influence contraction. Thus a major aim of this proposal is to clarify the role of protein kinase C on Ca2+ signalling in smooth muscle. We will approach this question by microinjecting protein kinase C modulators (specific inhibitory peptides, antibodies and functional domains of protein kinase C) into isolated single smooth muscle cells. These specific modulators of smooth muscle protein kinase C will be developed based upon studies which we propose using molecular cloning strategies to determine the structure of smooth muscle protein kinase C. The boundary of each functional domain will be analyzed by expressing and characterizing various mutants of recombinant proteins. This information will be directly used for developing modulated kinase probes, peptide probes and antibody probes. These probes will then be used in Project 1 to assess the role of protein kinase C in feedback control of Ca2+ signalling in single intact cells. The antibody probes will be utilized not only for modulating kinase activity but also for determining the 3D intracellular localization of each kinase isoform. The information obtained in Project 1 will be utilized to guide the search in this project for the target proteins of the kinases that are involved in the modulation of Ca2+ signals. Using a similar strategy, the role of calmodulin dependent protein kinase II on Ca2+ homeostatis will also be studied since our preliminary results suggest its involvement. These studies of physiological function of smooth muscle will provide important information into smooth muscle malfunction in diseases such as hyper- tension, asthma, and spastic disorders of gastrointestinal system.